Project Summary/Abstract While chronic pain represents a massive public health problem with a staggering economic cost of $560-$635 billion each year in the U.S. alone, the molecular mechanisms driving neuronal hyperexcitability within nociceptive pathways remain incompletely understood. Significant progress has been made towards elucidating the genetic heterogeneity of primary sensory neurons and their plasticity in the aftermath of nerve or tissue damage. However, much less is known about the comprehensive molecular profile of those neurons that convey nociceptive information from the spinal cord to the brain, despite their clear importance for pain perception. A better understanding of the complete pattern of gene expression within spinal projection neurons could reveal new evidence-based strategies to selectively dampen the output of the spinal nociceptive network as a means to alleviate chronic pain. The long-term goal is to better understand how nerve and tissue damage alter the function of nociceptive circuits in the CNS. The objective of this application is to identify injury-evoked changes in gene expression within spinal projection neurons that enhance their firing under chronic pain conditions. The central hypothesis is that ascending spinal projection neurons exhibit a unique molecular phenotype that is significantly modulated by peripheral injury to promote membrane hyperexcitability. The rationale for the proposed work is that the identification of genes that are preferentially expressed in spinal projection neurons will yield new pharmacological approaches to suppress the ascending flow of nociceptive information to the brain, while minimizing unwanted disruptions to global sensorimotor processing within the spinal cord. The central hypothesis will be tested by pursuing the following specific aims: (1) Identify genes that are enriched in ascending projection neurons within the adult spinal cord; and (2) Elucidate changes in gene expression in projection neurons under chronic pain conditions that increase membrane excitability. These aims will be accomplished by using translating ribosome affinity purification (TRAP) and next generation RNA sequencing techniques in combination with bioinformatics, electrophysiological, immunohistochemical and in situ hybridization approaches. The proposed work is innovative because it will reveal, for the first time, the genetic phenotype of those neurons connecting the spinal nociceptive circuit to the mouse brain that are critically involved in the generation of neuropathic and inflammatory pain, as well as elucidate how the molecular signature of this population changes during the chronic pain state. The outcome of these investigations will be the discovery of new, cell type-specific markers of spinal projection neurons and the identification of potential genetic mechanisms by which peripheral injuries can amplify the ?gain? of nociceptive transmission in the spinal cord. As a result, the proposed research is significant because it will reveal novel molecular targets which could be manipulated to selectively silence ascending spinal projection neurons after injury, in order to evoke safe and effective analgesia while minimizing undesirable side effects.